Miniaturized planar columns in novel support media for liquid phase analysis

ABSTRACT

Miniaturized planar column devices are described for use in liquid phase analysis, the devices comprising microstructures fabricated by laser ablation in a variety of novel support substrates. Devices formed according to the invention include associated laser-ablated features required for function, such as analyte detection means and fluid communication means. Miniaturized columns constructed under the invention find use in any analysis system performed on either small and/or macromolecular solutes in the liquid phase and may employ chromatographic, electrophoretic, electrochromatographic separation means, or any combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/824,620 filed on Mar.27, 1997, now U.S. Pat. No. 5,882,571 which is a division of U.S. patentapplication Ser. No. 08/482,245 filed on Jun. 7, 1995, now U.S. Pat. No.5,658,413, which is a continuation-in-part of U.S. patent applicationSer. No. 08/326,111, filed Oct. 19, 1994, now U.S. Pat. No. 5,500,071,from which priority is claimed pursuant to 35 U.S.C. §120, and whichdisclosure is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to miniaturized planar columntechnology for liquid phase analysis, and more particularly tofabrication of microstructures in novel separation support media usinglaser ablation techniques. The microstructures produced under thepresent invention find use in any analysis system which is performed oneither small and/or macromolecular solutes in the liquid phase and whichmay employ chromatographic or electrophoretic means of separation, or acombination of both.

BACKGROUND OF THE INVENTION

In sample analysis instrumentation, and especially in separation systemssuch as liquid chromatography and capillary electrophoresis systems,smaller dimensions will generally result in improved performancecharacteristics and at the same time result in reduced production andanalysis costs. In this regard, miniaturized separation systems providemore effective system design, result in lower overhead due to decreasedinstrumentation sizing and additionally enable increased speed ofanalysis, decreased sample and solvent consumption and the possibilityof increased detection efficiency.

Accordingly, several approaches towards miniaturization for liquid phaseanalysis have developed in the art; the conventional approach usingdrawn fused-silica capillary, and an evolving approach using siliconmicromachining. What is currently thought of as conventional inminiaturization technology is generally any step toward reduction insize of the analysis system.

In conventional miniaturized technology the instrumentation has not beenreduced in size; rather, it is the separation compartment size which hasbeen significantly reduced. As an example, micro-column liquidchromatography (μLC) has been described wherein columns with diametersof 100-200 μm are employed as compared to prior column diameters ofaround 4.6 mm.

Another approach towards miniaturization has been the use of capillaryelectrophoresis (CE) which entails a separation technique carried out incapillaries 25-100 μm in diameter. CE has been demonstrated to be usefulas a method for the separation of a variety of large and small solutes.J. Chromatog. 218:209 (1981); Analytical Chemistry 53:1298 (1981). Incontrast, polyacrylamide gel electrophoresis was originally carried outin tubes 1 mm in diameter. Both of the above described "conventional"miniaturization technologies (μLC and CE) represent a first significantstep toward reducing the size of the chemical portion of a liquid phaseanalytical system. However, even though experimentation with suchconventional miniaturized devices has helped to verify the advantages ofminiaturization in principal, there nevertheless remain several majorproblems inherent in those technologies.

For example, there remains substantial detection limitations inconventional capillary electrophoresis technology. For example, in CE,optical detection is generally performed on-column by a single-passdetection technique wherein electromagnetic energy is passed through thesample, the light beam travelling normal to the capillary axis andcrossing the capillary only a single time. Accordingly, in conventionalCE systems., the detection path length is inherently limited by thediameter of the capillary.

Given Beer's law, which relates absorbance to the path length throughthe following relationship:

    A=ε*b*C

where:

A=the absorbance

ε=the molar absorptivity, (l/m*cm)

b=pathlength (cm)

C=concentration (m/l)

it can be readily understood that the absorbance (A) of a sample in a 25μm capillary would be a factor of 400× less than it would be in aconventional 1 cm pathlength cell as typically used in UV/Visspectroscopy.

In light of this significant detection limitation, there have been anumber of attempts employed in the prior art to extend detection pathlengths, and hence the sensitivity of the analysis in CE systems. InU.S. Pat. No. 5,061,361 to Gordon, there has been described an approachentailing micro-manipulation of the capillary flow-cell to form a bubbleat the point of detection. In U.S. Pat. No. 5,141,548 to Chervet, theuse of a Z-shaped configuration in the capillary, with detectionperformed across the extended portion of the Z has been described. Yetanother approach has sought to increase the detection pathlength bydetecting along the major axis of the capillary (axial-beam detection).Xi et al., Analytical Chemistry 62:1580 (1990).

In U.S. Pat. No. 5,273,633 to Wang, a further approach to increaseddetection path lengths in CE has been described where a reflectingsurface exterior of the capillary is provided, the subject systemfurther including an incident window and an exit window downstream ofthe incident window. Under Wang, light entering the incident windowpasses through a section of the capillary by multiple internalreflections before passing through the exit window where it is detected,the subject multiple internal reflections yielding an effective increasein pathlength. While each of the aforementioned approaches has addressedthe issue of extending the pathlength, each approach is limited in thatit entails engineering the capillary after-the-fact or otherwiseincreasing the cost of the analysis.

A second major drawback in the current approach to miniaturizationinvolves the chemical activity and chemical instability of silicondioxide (SiO₂) substrates, such as silica, quartz or glass, which arecommonly used in both CE and μLC systems. More particularly, silicondioxide substrates are characterized as high energy surfaces andstrongly adsorb many compounds, most notably bases. The use of silicondioxide materials in separation systems is further restricted due to thechemical instability of those substrates, as the dissolution of SiO₂materials increases in basic conditions (at pHs greater than 7.0).

To avoid the problems arising from the inherent chemical activity ofsilicon dioxide materials, prior separation systems have attemptedchemical modifications to the inner silica surface of capillary walls.In general, such post-formation modifications are difficult as theyrequire the provision of an interfacial layer to bond a desired surfacetreatment to the capillary surface, using, for example, silylatingagents to create Si--O--Si--C bonds. Although such modifications maydecrease the irreversible adsorption of solute molecules by thecapillary surfaces, these systems still suffer from the chemicalinstability of Si--O--Si bonds at pH's above 7.0. Accordingly, chemicalinstability in SiO₂ materials remains a major problem.

However, despite the recognized shortcomings with the chemistry of SiO₂substrates, those materials are still used in separation systems due totheir desirable optical properties. In this regard, potential substitutematerials which exhibit superior chemical properties compared to silicondioxide materials are generally limited in that they are also highlyadsorbing in the UV region, where detection is important.

In order to avoid some of the substantial limitations present inconventional μLC and CE techniques, and in order to enable even greaterreduction in separation system sizes, there has been a trend towardsproviding planarized systems having capillary separationmicrostructures. In this regard, production of miniaturized separationsystems involving fabrication of microstructures in silicon bymicromachining or microlithographic techniques has been described. See,e.g. Fan et al., Anal. Chem. 66(1):177-184 (1994); Manz et al., Adv. inChrom. 33:1-66 (1993); Harrison et al., Sens. Actuators, BB10(2):107-116 (1993); Manz et al., Trends Anal. Chem. 10(5):144-149(1991); and Manz et al., Sensors and Actuators B (Chemical)B1(1-6):249-255 (1990).

The use of micromachining techniques to fabricate separation systems insilicon provides the practical benefit of enabling mass production ofsuch systems. In this regard, a number of established techniquesdeveloped by the microelectronics industry involving micromachining ofplanar materials, such as silicon, exist and provide a useful and wellaccepted approach to miniaturization. Examples of the use of suchmicromachining techniques to produce miniaturized separation devices onsilicon or borosilicate glass chips can be found in U.S. Pat. No.5,194,133 to Clark et al.; U.S. Pat. No. 5,132,012 to Miura et al.; inU.S. Pat. No. 4,908,112 to Pace; and in U.S. Pat. No. 4,891,120 to Sethiet al.

Micromachining silicon substrates to form miniaturized separationsystems generally involves a combination of film deposition,photolithography, etching and bonding techniques to fabricate a widearray of three dimensional structures. Silicon provides a usefulsubstrate in this regard since it exhibits high strength and hardnesscharacteristics and can be micromachined to provide structures havingdimensions in the order of a few micrometers.

Although silicon micromachining has been useful in the fabrication ofminiaturized systems on a single surface, there are significantdisadvantages to the use of this approach in creating the analysisdevice portion of a miniaturized separation system.

Initially, silicon micromachining is not amenable to producing a highdegree of alignment between two etched or machined pieces. This has anegative impact on the symmetry and shape of a separation channel formedby micromachining, which in turn may impact separation efficiency.Secondly, sealing of micromachined silicon surfaces is generally carriedout using adhesives which may be prone to attack by separationconditions imposed by liquid phase analyses. Furthermore, underoxidizing conditions, a silica surface is formed on the silicon chipsubstrate. Thus, silicon micromachining is also fundamentally limited bythe chemistry of SiO₂. Accordingly, there has remained a need for animproved miniaturized separation system which is able to avoid theinherent shortcomings of conventional miniaturization and siliconmicromachining techniques.

SUMMARY OF THE INVENTION

The present invention relates to a miniaturized planar column device foruse in a liquid phase analysis system. It is a primary object of theinvention to provide a miniaturized column device laser-ablated in asubstantially planar substrate, wherein the substrate is a materialselected to avoid the inherent chemical activity and pH instabilityencountered with silicon and prior silicon dioxide-based devicesubstrates.

The invention is also related to the provision of detection meansengineered into a miniaturized planar column device whereby enhancedon-column analysis or detection of components in a liquid sample isenabled. It is a related object of the invention to provide a columndevice for liquid phase analysis having detection means designed intothe device in significantly compact form as compared to conventionaltechnology. It is a further object to provide optical detection meansablated in a miniaturized planar column device and having asubstantially enhanced detection pathlength. It is yet a further objectto provide a plurality of detection means which allow simultaneousinterrogation of a liquid sample to detect separated analytes usingmultiple detection techniques that communicate with the sample at aparticular position along the separation compartment.

In another aspect of the invention a device is provided which featuresimproved means for liquid handling, including sample injection. In arelated aspect, a miniaturized column device is provided having a meansto interface with a variety of external liquid reservoirs. In aparticular embodiment, a system design is provided which allows avariety of injection methods to be readily adapted to the planarstructure, such as pressure injection, hydrodynamic injection orelectrokinetic injection.

It is yet a further related object of the invention to provide aminiaturized total chemical analysis system (μ-TAS) fully contained on asingle, planar surface. Particularly, a miniaturized system according tothe present invention is capable of performing complex sample handling,separation, and detection methods with reduced technician manipulationor interaction. Thus, the subject invention finds potential applicationin monitoring and/or analysis of components in industrial chemical,biological, biochemical and medical processes and the like.

A particular advantage provided by the invention is the use of processesother than silicon micromachining techniques or etching techniques tocreate miniaturized columns in a wide variety of polymeric and ceramicsubstrates having desirable attributes for an analysis portion of aseparation system. More specifically, a miniaturized planar columndevice is formed herein by ablating component microstructures in asubstrate using laser radiation. In one preferred embodiment, aminiaturized column device is formed by providing two substantiallyplanar halves having microstructures laser-ablated thereon, which, whenthe two halves are folded upon each other, define a separationcompartment featuring enhanced symmetry and axial alignment.

Use of laser ablation techniques to form miniaturized devices accordingto the invention affords several advantages over prior etching andmicromachining techniques used to form systems in silicon or silicondioxide materials. Initially, the capability of applying rigidcomputerized control over laser ablation processes allows microstructureformation to be executed with great precision, thereby enabling aheightened degree of alignment in structures formed by component parts.The laser ablation process also avoids problems encountered withmicrolithographic isotropic etching techniques which may undercutmasking during etching, giving rise to asymmetrical structures havingcurved side walls and flat bottoms.

Laser ablation further enables the creation of microstructures withgreatly reduced component size. In this regard, microstructures formedaccording to the invention are capable of having aspect ratios severalorders of magnitude higher than possible using prior etching techniques,thereby providing enhanced separation capabilities in such devices. Theuse of laser-ablation processes to form microstructures in substratessuch as polymers increases ease of fabrication and lowers per-unitmanufacturing costs in the subject devices as compared to priorapproaches such as micromachining devices in silicon. Devices formedunder the invention in low-cost polymer substrates have the addedfeature of being capable of use as substantially disposable miniaturizedcolumn units.

In another aspect of the invention, laser-ablation in planar substratesallows for the formation of microstructures of almost any geometry orshape. This feature not only enables the formation of complex deviceconfigurations, but further allows for integration of samplepreparation, sample injection, post-column reaction and detection meansin a miniaturized total analysis system of greatly reduced overalldimensions.

The compactness of the analysis portion in a device formed herein--inconjunction with the feature that integral functions such as injection,sample handling and detection may be specifically engineered into thesubject device to provide a μ-TAS device--further allows for integrateddesign of system hardware to achieve a greatly reduced system footprint.

Thus, inherent weaknesses existing in prior approaches to liquid phaseseparation device miniaturization, and problems in using siliconmicromachining techniques to form miniaturized column devices have beenaddressed. Accordingly, the present invention discloses a miniaturizedcolumn device capable of performing a variety of liquid phase analyseson a wide array of liquid samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exploded view of a miniaturized column device constructedin accordance with the invention.

FIG. 1B is an exploded view of a preferred embodiment of the presentinvention including optical detection means.

FIG. 2 is a plan view of the interior surface of the miniaturized columndevice of FIG. 1A.

FIG. 3 is a plan view of the exterior surface of the device of FIG. 1A.

FIG. 4 is a cross-sectional side view of the miniaturized column deviceof FIG. 1A, taken along lines IV--IV and showing formation of aseparation compartment according to the invention.

FIG. 5 is a plan view of a preferred embodiment of the miniaturizedcolumn device of FIG. 1A which is constructed from a single flexiblesubstrate.

FIG. 6 is a cross-sectional axial view of the intersection of theseparation compartment and the optical detection means in theminiaturized column device of FIG. 1B.

FIG. 7A is an exploded view of a first side of a miniaturized columndevice having microchannels formed on two opposing planar surfaces of asupport substrate.

FIG. 7B is an exploded view of a second side of the column device ofFIG. 7A.

FIG. 8A is a pictorial representation of a first side of a preferredembodiment of the miniaturized column device of FIG. 7A which isconstructed from a single flexible substrate.

FIG. 8B is a pictorial representation of a second side of the columndevice of FIG. 8A.

FIG. 9 is a cross-sectional trans-axial view of the extended opticaldetection pathlength in the miniaturized column of FIG. 7B taken alonglines IX--IX.

FIG. 10 is plan view of a miniaturized column device constructed underthe invention having first and second component halves.

FIG. 11 is a pictorial representation of the column device of FIG. 10showing the folding alignment of the component halves to form a singledevice.

FIG. 12 is a cross-sectional axial view of the separation compartmentformed by the alignment of the component halves in the device of FIG.10.

FIG. 13 is a plan view of a further preferred embodiment of the presentinvention having optional micro-alignment means on first and secondcomponent halves.

FIG. 14 is a pictorial representation of the column device of FIG. 13showing the micro-alignment of the component halves.

FIG. 15 is a plan view of the top surface of a miniaturized columndevice including electrical communication path detection means arrangedopposite each other relative to a separation compartment.

FIG. 16 is a pictorial representation of the electrode means of FIG. 15.

FIG. 17 is a pictorial representation of the electrode means depicted inFIG. 16 showing a preferred height feature thereof.

FIG. 18 is a pictorial representation of the top surface of aminiaturized column device including electrode means arrangedsubstantially parallel relative each other on a first side of aseparation compartment.

FIG. 19 is a pictorial representation of the electrode means of FIG. 18.

FIG. 20 is a pictorial representation of the electrode means depicted inFIG. 19 showing a preferred height feature thereof.

FIG. 21 is a pictorial representation of the top surface of aminiaturized column device including a plurality of serially arrangedannular electrode coils.

FIG. 22 is a pictorial representation similar to FIG. 21 wherein thecover plate is removed to expose the annular electrode coils.

FIG. 23 is a pictorial representation demonstrating a preferred methodof forming the electrodes of FIG. 22.

FIG. 24 is a pictorial representation of the annular electrodes of FIG.22 illustrating the coaxial-arrangement thereof about a separationcompartment.

FIG. 25 is a cross-sectional view of an annular electrode coil from FIG.22.

FIG. 26 is an exploded view of a miniaturized column device having anassociated lightguide means disposed within a detection means.

FIG. 27 is a pictorial representation of the optional lightguide meanscommunicating with the separation compartment of the device of FIG. 26.

FIG. 28 is an exploded view of a miniaturized column device having aplurality of associated lightguide means.

FIG. 29 is a pictorial representation of the plurality of lightguidemeans communicating with the separation compartment of the device ofFIG. 28.

FIG. 30 is a pictorial representation of a miniaturized column devicehaving a detection intersection formed by two orthogonal detectionpaths.

FIG. 31 is a pictorial representation of a miniaturized column devicehaving a detection intersection formed by a detection path and a furtherdetection means arranged in orthogonal relation to each other.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the invention is described in detail, it is to be understood thatthis invention is not limited to the particular component parts of thedevices described or process steps of the methods described as suchdevices and methods may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms "a," "an" and "the" include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to "an analyte"includes mixtures of analytes, reference to "a detection means" includestwo or more such detection means, and the like.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

The term "substrate" is used herein to refer to any material which isUV-adsorbing, capable of being laser-ablated and which is not silicon ora silicon dioxide material such as quartz, fused silica or glass(borosilicates). Accordingly, miniaturized column devices are formedherein using suitable substrates, such as laser ablatable polymers(including polyimides and the like) and ceramics (including aluminumoxides and the like). Further, miniaturized column devices are formedherein using composite substrates such as laminates. A "laminate" refersto a composite material formed from several different bonded layers ofsame or different materials. One particularly preferred compositesubstrate comprises a polyimide laminate formed from a first layer ofpolyimide such as Kapton®, available from DuPont (Wilmington, Del.),that has been co-extruded with a second, thin layer of a thermaladhesive form of polyimide known as KJ® available from DuPont(Wilmington, Del.). This thermoplastic adhesive layer can be on one orboth sides of the first polyimide layer, thereby providing a laminatestructure of any desired thickness.

As used herein, the term "detection means" refers to any means,structure or configuration which allows one to interrogate a samplewithin the separation compartment using analytical detection techniqueswell known in the art. Thus, a detection means includes one or moreapertures, elongated apertures or grooves which communicate with theseparation compartment and allow an external detection apparatus ordevice to be interfaced with the separation compartment to detect ananalyte passing through the compartment.

Changes in the electrochemical properties of a liquid sample passingthrough the separation compartment can be detected using detection meanswhich physically contact the sample passing through the separationcompartment. In one embodiment, an electrode may be placed within, orbutt-coupled to a detection means such as an aperture or a groove,thereby enabling the electrode to directly contact the sample stream. Byarranging two dissimilar electrodes (which are connected through anexternal conducting circuit) opposite each other relative to theseparation compartment, an electric field can be generated in theseparation compartment--transverse to the direction of sampleflow--thereby providing a ready means of electrochemical detection ofanalytes passing through the compartment.

Changes in the electrical properties of a liquid sample passing throughthe separation compartment can be detected using detection means whichdo not directly contact the sample passing through the separationcompartment. Thus, "changes in the electrical properties" of a samplepassing through the separation compartment refers to detectable changesin the conductivity, permittivity, or both of a particular sample due tothe presence of an analyte in the sample. The "conductivity" of a samplerefers to the ratio of the electric current density to the electricfield in that sample. The "permittivity" of a sample refers to thedielectric constant of a sample multiplied by the permittivity of emptyspace, where the permittivity of empty space (ε₀) is a constantappearing in Coulomb's law having the value of 1 incentimeter-gram-second electrostatic units.

Changes in the electrical properties of a sample passing through aseparation compartment are measured herein by detection of the impedanceof the liquid sample. The "impedance" or "electrical impedance" of acircuit refers to the total opposition that the circuit presents to analternating current ("AC"), equal to the complex ratio of the voltage tothe current in complex notation. Thus, the magnitude of the totalopposition that a circuit presents to an alternating current is equal tothe ratio of the maximum voltage in an AC circuit to the maximumcurrent. An "electrical impedance meter" refers to an instrument whichmeasures the complex ratio of voltage to current in a given circuit at agiven frequency.

A plurality of electrical "communication paths" capable of carryingand/or transmitting electric current can be arranged adjacent to theseparation compartment such that the communication paths, incombination, form a circuit. As used herein, a communication pathincludes any conductive material which is capable of transmitting orreceiving an AC signal. A particularly preferred conductive material iscopper. Thus, in one embodiment, a plurality of communication pathsforming an antenna circuit (e.g., a pair of copper antennae) arearranged adjacent to the separation compartment whereby a circuit isformed capable of passing an oscillating voltage through the separationcompartment which is sensitive to changes in the impedance of a liquidsample flowing therethrough. An "antenna" refers to a device capable ofradiating and/or receiving radio waves such as an alternating current(AC) signal. An "antenna circuit" intends a complete electrical circuitwhich includes an antenna. An "antenna coil" refers to a coil throughwhich antenna current (e.g., an AC signal) flows.

Further, by the arrangement of two detection means opposite each otherrelative to the separation compartment, a "detection path" isconveniently formed, thereby allowing detection of analytes passingthrough the separation compartment using detection techniques well knownin the art.

An "optical detection path" refers to a configuration or arrangement ofdetection means to form a path whereby electromagnetic radiation is ableto travel from an external source to a means for receivingradiation--wherein the radiation traverses the separation compartmentand can be influenced by the sample or separated analytes in the sampleflowing through the separation compartment. An optical detection path isgenerally formed under the invention by positioning a pair of detectionmeans directly opposite each other relative to the separationcompartment. In this configuration, analytes passing through theseparation compartment can be detected via transmission of radiationorthogonal to the major axis of the separation compartment (and,accordingly, orthogonal to the direction of electro-osmotic flow in anelectrophoretic separation). A variety of external optical detectiontechniques can be readily interfaced with the separation compartmentusing an optical detection path including, but not limited to, UV/Vis,Near IR, fluorescence, refractive index (RI) and Raman techniques.

As used herein, the term "transparent" refers to the ability of asubstance to transmit light of different wavelengths, which ability maybe measured in a particular substance as the percent of radiation whichpenetrates a distance of 1 meter. Thus, under the invention, a"transparent sheet" is defined as a sheet of a substance which istransmissive to specific types of radiation or particles of interest.Transparent sheets which are particularly employed in the invention inthe context of optical detection configurations are formed frommaterials such as, but not limited to, quartz, sapphire, diamond andfused silica.

In the context of UV-visible absorption detection of sample analytesherein, the terms "path length," or "optical path length" refer to anoptical path length "b" derived from Beer's law, which states that

    A=log(I.sub.i /I.sub.f)=ε*b*C

wherein A is the absorbance, I_(i) is the light intensity measured inthe absence of the analyte, I_(f) is the light intensity transmittedthrough the analyte, ε is the molar extinction coefficient of the sample(l/m*cm), C is the analyte concentration (m/l), and b is the opticalpathlength (cm). Thus, in a detection configuration wherein UV-visabsorption of a sample analyte is measured via an optical detection pathby passing light through the separation compartment along a pathperpendicular to the separation compartment major axis, the path length(b) of the measurement is substantially defined by the dimensions of theseparation compartment.

A "detection intersection" refers to a configuration wherein a pluralityof detection means that communicate with the separation compartmentconverge at a particular location in the separation compartment. In thismanner, a number of detection techniques can be simultaneously performedon a sample or separated analyte at the detection intersection. Underthe invention, a detection intersection is formed when a plurality ofdetection paths cross, or when a detection means such as an aperturecommunicates with the separation compartment at substantially the samepoint as a detection path. The sample, or a separated analyte, can thusbe interrogated using a combination of UV/Vis and fluorescencetechniques, optical and electrochemical techniques, optical andelectrical techniques, or like combinations to provide highly sensitivedetection information. See, e.g., Beckers et al. (1988) J. Chromatogr.452:591-600; and U.S. Pat. No. 4,927,265, to Brownlee.

As used herein, a "lightguide means" refers to a substantially long,thin thread of a transparent substance which can be used to transmitlight. Lightguide means useful in the practice of the invention includeoptical fibers, integrated lens configurations and the like. Inparticularly preferred embodiments, optical fibers are interfaced withdetection means to enable optical detection techniques known in the art.The terms "optical fiber," "fiber optic waveguide" or "optical fibermeans" are used herein to refer to a single optical fiber or a bundleoptical fibers, optionally encased in a protective cladding material.Examples of suitable optical fiber substrate materials include glass,plastic, glass/glass composite and glass/plastic composite fibers. Acritical characteristic of optical fibers is attenuation of an opticalsignal. Further, a chemical sensor can be incorporated into a fiberoptic waveguide in a manner such that the chemical sensor will interactwith the liquid sample analyte. Structures, properties, functions andoperational details of such fiber optic chemical sensors can be found inU.S. Pat. No. 4,577,109 to Hirschfeld, U.S. Pat. No. 4,785,814 to Kane,and U.S. Pat. No. 4,842,783 to Blaylock.

The use of laser ablation techniques in the practice of the inventionallows for a high degree of precision in the alignment of micro-scalecomponents and structures, which alignment has either been difficult ornot possible in prior silicon or glass substrate-based devices. Thus,the term "microalignment" as used herein refers to the precise andaccurate alignment of laser-ablated features, including the enhancedalignment of complementary microchannels or microcompartments with eachother, inlet and/or outlet ports with microchannels or separationcompartments, detection means with microchannels or separationcompartments, detection means with other detection means, and the like.

The term "microalignment means" is defined herein to refer to any meansfor ensuring the precise microalignment of laser-ablated features in aminiaturized column device. Microalignment means can be formed in thecolumn devices either by laser ablation or other methods of fabricatingshaped pieces, which methods are well known in the art. Representativemicro-alignment means that can be employed herein include a plurality ofcoaxially arranged apertures laser-ablated in component parts and/or aplurality of corresponding features in column device substrates, e.g.,projections and mating depressions, grooves and mating ridges or thelike. Further, the accurate microalignment of component parts can beeffected by forming the miniaturized columns in flexible substrateshaving at least one fold means laser-ablated therein, such that sectionsof the substrate can be folded to overlie other sections thereby formingcomposite micro-scale compartments, aligning features such as aperturesor detection means with separation compartments, or forming micro-scaleseparation compartments from microchannels. Such fold means can beembodied by a row of spaced-apart perforations ablated in a particularsubstrate, spaced-apart slot-like depressions or apertures ablated so asto extend only part way through the substrate. The perforations ordepressions can have circular, diamond, hexagonal or other shapes thatpromote hinge formation along a predetermined straight line.

The term "liquid phase analysis" is used to refer to any analysis whichis done on either small and/or macromolecular solutes in the liquidphase. Accordingly, "liquid phase analysis" as used herein includeschromatographic separations, electrophoretic separations, andelectrochromatographic separations.

In this regard, "chromatographic" processes generally comprisepreferential separations of components, and include reverse-phase,hydrophobic interaction, ion exchange, molecular sieve chromatographyand like methods.

"Electrophoretic" separations refers to the migration of particles ormacromolecules having a net electric charge where said migration isinfluenced by an electric field. Accordingly electrophoretic separationsinclude separations performed in columns packed with gels (such aspoly-acrylamide, agarose and combinations thereof) as well asseparations performed in solution.

"Electrochromatographic" separations refers to combinations ofelectrophoretic and chromatographic techniques. Exemplaryelectrochromatographic separations include packed column separationsusing electromotive force (Knox et al. (1987) Chromatographia 24:135;Knox et al. (1989) J. Liq. Chromatogr 12:2435; Knox et al. (1991)Chromatographia 32:317), and micellar electrophoretic separations(Terabe et al. (1985) Anal. Chem. 57:834-841).

The term "motive force" is used to refer to any means for inducingmovement of a sample along a column in a liquid phase analysis, andincludes application of an electric potential across any portion of thecolumn, application of a pressure differential across any portion of thecolumn or any combination thereof.

The term "surface treatment" is used to refer to preparation ormodification of the surface of a microchannel which will be in contactwith a sample during separation, whereby the separation characteristicsof the device are altered or otherwise enhanced. Accordingly, "surfacetreatment" as used herein includes: physical surface adsorptions;covalent bonding of selected moieties to functional groups on thesurface of microchannel substrates (such as to amine, hydroxyl orcarboxylic acid groups on condensation polymers); methods of coatingsurfaces, including dynamic deactivation of channel surfaces (such as byadding surfactants to media), polymer grafting to the surface of channelsubstrates (such as polystyrene or divinyl-benzene) and thin-filmdeposition of materials such as diamond or sapphire to microchannelsubstrates.

The term "laser ablation" is used to refer to a machining process usinga high-energy photon laser such as an excimer laser to ablate featuresin a suitable substrate. The excimer laser can be, for example, of theF₂, ArF, KrCl, KrF, or XeCl type.

In general, any substrate which is UV absorbing provides a suitablesubstrate in which one can laser ablate features. Accordingly,microstructures of selected configurations can be formed by imaging alithographic mask onto a suitable substrate, such as a polymer orceramic material, and then laser ablating the substrate with laser lightin areas that are unprotected by the lithographic mask.

In laser ablation, short pulses of intense ultraviolet light areabsorbed in a thin surface layer of material within about 1 μm or lessof the surface. Preferred pulse energies are greater than about 100millijoules per square centimeter and pulse durations are shorter thanabout 1 microsecond. Under these conditions, the intense ultravioletlight photo-dissociates the chemical bonds in the material. Furthermore,the absorbed ultraviolet energy is concentrated in such a small volumeof material that it rapidly heats the dissociated fragments and ejectsthem away from the surface of the material. Because these processesoccur so quickly, there is no time for heat to propagate to thesurrounding material. As a result, the surrounding region is not meltedor otherwise damaged, and the perimeter of ablated features canreplicate the shape of the incident optical beam with precision on thescale of about one micrometer.

Although laser ablation has been described herein using an excimerlaser, it is to be understood that other ultraviolet light sources withsubstantially the same optical wavelength and energy density may be usedto accomplish the ablation process. Preferably, the wavelength of suchan ultraviolet light source will lie in the 150 nm to 400 nm range toallow high absorption in the substrate to be ablated. Furthermore, theenergy density should be greater than about 100 millijoules per squarecentimeter with a pulse length shorter than about 1 microsecond toachieve rapid ejection of ablated material with essentially no heatingof the surrounding remaining material. Laser ablation techniques, suchas those described above, have been described in the art. Znotins, T.A., et al., Laser Focus Electro Optics, (1987) pp. 54-70; U.S. Pat. Nos.5,291,226 and 5,305,015 to Schantz et al.

The term "injection molding" is used to refer to a process for moldingplastic or nonplastic ceramic shapes by injecting a measured quantity ofa molten plastic or ceramic substrate into dies (or molds). In oneembodiment of the present invention, miniaturized column devices can beproduced using injection molding.

More particularly, a mold or die of a miniaturized column device isformed using excimer laser-ablation to define an original microstructurepattern in a suitable polymer substrate. The microstructure thus formedis then coated by a very thin metal layer and electroplated (such as bygalvano forming) with a metal such as nickel to provide a carrier. Whenthe metal carrier is separated from the original polymer, a mold insert(or tooling) is provided having the negative structure of the polymer.Multiple replicas of the ablated microstructure pattern can thus bemanufactured in suitable polymer or ceramic substrates using injectionmolding techniques well known in the art.

The term "LIGA process" is used to refer to a process for fabricatingmicrostructures having high aspect ratios and increased structuralprecision using synchrotron radiation lithography, galvanoforming, andplastic molding. In a LIGA process, radiation sensitive plastics arelithographically irradiated at high energy radiation using a synchrotronsource to create desired microstructures (such as channels, ports,apertures and micro-alignment means), thereby forming a primarytemplate.

The primary template is then filled with a metal by electrodepositiontechniques. The metal structure thus formed comprises a mold insert forthe fabrication of secondary plastic templates which take the place ofthe primary template. In this manner, highly accurate replicas of theoriginal microstructures can be formed in a variety of substrates usinginjection or reactive injection molding techniques. The LIGA process hasbeen described by Becker, E. W., et al., Microelectric Engineering 4(1986) pp. 35-56. Descriptions of numerous polymer substrates which canbe injection molded using LIGA templates, and which are suitablesubstrates in the practice of the subject invention, may be found in"Contemporary Polymer Chemistry", Allcock, H. R. and Lampe, F. W.(Prentice-Hall, Inc.) New Jersey (1981).

Accordingly, the invention concerns formation of miniaturized columndevices using laser ablation in a suitable substrate. The column devicesare also formed using injection molding techniques wherein the originalmicrostructure has been formed by an excimer laser ablation process, orwhere the original microstructure has been formed using a LIGA process.

More particularly, microstructures such as separation compartments,injection means, detection means and micro-alignment means can be formedin a planar substrate by excimer laser ablation. A frequency multipliedYAG laser can also be used in place of the excimer laser. In such acase, a complex microstructure pattern useful for practicing theinvention can be formed on a suitable polymeric or ceramic substrate bycombining a masking process with a laser ablation means, such as in astep-and-repeat process, where such processes would be readilyunderstood by one of ordinary skill in the art.

In the practice of the invention, a preferred substrate comprises apolyimide material such as those available under the trademarks Kapton®or Upilex® from DuPont (Wilmington, Del.), although the particularsubstrate selected can comprise any other suitable polymer or ceramicsubstrate. Polymer materials particularly contemplated herein includematerials selected from the following classes: polyimide, polycarbonate,polyester, polyamide, polyether, polyolefin, or mixtures thereof.Further, the polymer material selected can be produced in long strips ona reel, and, optional sprocket holes along the sides of the material maybe provided to accurately and securely transport the substrate through astep-and-repeat process.

The selected polymer material is transported to a laser processingchamber and laser-ablated in a pattern defined by one or more masksusing laser radiation. In a preferred embodiment, such masks define allof the ablated features for an extended area of the material, forexample encompassing multiple apertures (including inlet and outletports), micro-alignment means and separation chambers.

Alternatively, patterns such as the aperture pattern, the separationchannel pattern, etc., can be placed side by side on a common masksubstrate which is substantially larger than the laser beam. Suchpatterns are then moved sequentially into the beam. In other productionmethods, one or more masks can be used to form apertures through thesubstrate, and another mask and laser energy level (and/or number oflaser shots) can be used to define separation channels which are onlyformed through a portion of the thickness of the substrate. The maskingmaterial used in such masks is, preferably, highly reflecting at thelaser wavelength, consisting of, for example, a multilayer dielectricmaterial or a metal such as aluminum.

The laser ablation system employed in the invention generally includesbeam delivery optics, alignment optics, a high precision and high speedmask shuttle system, and a processing chamber including mechanism forhandling and positioning the material. In a preferred embodiment, thelaser system uses a projection mask configuration wherein a precisionlens interposed between the mask and the substrate projects the excimerlaser light onto the substrate in the image of the pattern defined onthe mask.

It will be readily apparent to one of ordinary skill in the art thatlaser ablation can be used to form miniaturized separation channels andapertures in a wide variety of geometries. Any geometry which does notinclude undercutting can be provided using ablation techniques, such asmodulation of laser light intensity across the substrate, stepping thebeam across the surface or stepping the fluence and number of pulsesapplied to each location to control corresponding depth. Further,laser-ablated channels or chambers produced according to the inventionare easily fabricated having ratios of channel depth to channel widthwhich are much greater than previously possible using etching techniquessuch as silicon micromachining. Such aspect ratios can easily exceedunity, and may even reach to 10.

In a preferred embodiment of the invention, channels of a semi-circularcross-section are laser ablated by controlling exposure intensity or bymaking multiple exposures with the beam being reoriented between eachexposure. Accordingly, when a corresponding semicircular channel isaligned with a channel thus formed, a separation chamber of highlysymmetrical circular cross-section is defined which may be desirable forenhanced fluid flow through the separation device.

As a final step in laser ablation processes contemplated by theinvention, a cleaning step is performed wherein the laser-ablatedportion of the substrate is positioned under a cleaning station. At thecleaning station, debris from the laser ablation are removed accordingto standard industry practice.

"Optional" or "optionally" as used herein means that the subsequentlydescribed feature or structure may or may not be present on theminiaturized column device, or that the subsequently described event orcircumstance may or may not occur; and that the description includesinstances where a particular feature or structure is present andinstances where the feature or structure is absent, or instances wherethe event or circumstance occurs and instances where it does not. Forexample, the phrase "a column device optionally having microalignmentmeans" intends that microalignment means may or may not be present onthe device and that the description includes both circumstances wheresuch means are present and absent.

As will be appreciated by those working in the field of liquid phaseanalysis devices, the above-described method can be used to produce awide variety of miniaturized devices. One such device is represented inFIG. 1A where a particular embodiment of a miniaturized column device isgenerally indicated at 2. The miniaturized column 2 is formed in aselected substrate 4 using laser ablation techniques. The substrate 4generally comprises first and second substantially planar opposingsurfaces indicated at 6 and 8 respectively, and is selected from amaterial other than silicon which is UV absorbing and, accordingly,laser-ablatable.

In a particular embodiment of the invention, the miniaturized columndevice 2 comprises a column structure ablated on a chip, which, in thepractice of the invention may be a machinable form of the plasticpolyimide such as Vespel®. The use of this particular polyimidesubstrate is preferred as, based on considerable experience with theshortcomings of fused silica and research into alternatives thereof,polyimides have proved to be a highly desirable substrate material forthe analysis portion of a liquid phase separation system.

In this regard, it has been demonstrated that polyimides exhibit lowsorptive properties towards proteins, which are known to be particularlydifficult to analyze in prior silicon dioxide-based separation systems.Successful demonstrations of separations with this difficult class ofsolutes typically ensures that separation of other classes of soluteswill be not be problematic. Further, since polyimide is a condensationpolymer, it is possible to chemically bond groups to the surface whichcan provide a variety of desirable surface properties, depending on thetarget analysis. Unlike prior silicon dioxide based systems, these bondsto the polymeric substrate demonstrate pH stability in the basic region(pH 9-10).

Referring now to FIGS. 1A, 2 and 3, the substrate 4 has a microchannel10 laser-ablated in a first planar surface 6. It will be readilyappreciated that, although the microchannel 10 has been represented in agenerally extended form, microchannels formed under the invention can beablated in a large variety of configurations, such as in a straight,serpentine, spiral, or any tortuous path desired. Further, as describedabove, the microchannel 10 can be formed in a wide variety of channelgeometries including semicircular, rectangular, rhomboid, and the like,and the channels can be formed in a wide range of aspect ratios. It isalso noted that a device having a plurality of microchannelslaser-ablated thereon falls within the spirit of the invention.

Referring particularly to FIGS. 1A and 4, a cover plate 12 is arrangedover the first planar surface 6 and, in combination with thelaser-ablated microchannel 10, forms an elongate separation compartment14. The cover plate 12 can be formed from any suitable substrate such aspolyimide, where the selection of the substrate is limited only byavoidance of undesirable separation surfaces such as silicon or silicondioxide materials.

In various embodiments, the cover plate 12 can be fixably aligned overthe first planar surface 6 to form a liquid-tight separation compartmentby using pressure sealing techniques, by using external means to urgethe pieces together (such as clips, tension springs or associatedclamping apparatus) or by using adhesives well known in the art ofbonding polymers, ceramics and the like.

Referring to FIGS. 1A and 2-4, a particular embodiment of the inventionis shown wherein the cover plate 12 further includes apertures whichhave been ablated therein. Particularly, a first aperture communicateswith the separation compartment 14--which has been formed by thecombination of microchannel 10 and cover plate 12--at a first end 16thereof to form an inlet port 18 enabling the passage of fluid from anexternal source into the separation compartment. A second aperturecommunicates with the separation compartment 14 at a second end 20thereof to form an outlet port 22 enabling passage of fluid from theseparation compartment to an external receptacle. Accordingly, aminiaturized column device is formed having a flow path extending fromthe first end 16 of the separation compartment and passing to the secondend 20 thereof, whereby liquid phase analysis of samples can be carriedout using techniques well known in the art.

Referring still to FIGS. 1A and 2-4, a particular embodiment of theinvention is shown including sample introduction means laser-ablatedinto both the substrate 4 and cover plate 12. An internally ablatedby-pass channel 24 is formed in substrate 4, such that the channel 24 isdisposed near the first end 16 of the separation compartment. Twoadditional apertures 26 and 28 are formed in cover plate 12 and arearranged to cooperate with first and second ends (indicated at 30 and 32respectively) of the by-pass channel 24. In this manner, a sample beingheld in an external reservoir can be introduced into by-pass channel 24to form a sample plug of a known volume (defined by the dimensions ofthe channel 24). The sample plug thus formed can then be introduced intothe first end 16 of the separation compartment 14 via inlet port 18 bycommunicating external mechanical valving with the inlet port andlaser-ablated apertures 26 and 28 and flushing solution through theby-pass channel 24 into the separation compartment.

It is noted that the ablated by-pass channel 24 and apertures 26 and 28further enable a wide variety of sample introduction techniques to bepracticed. Particularly, having a by-pass channel which is not connectedto the separation compartment allows a user to flush a sample throughthe by-pass channel without experiencing sample carry-over or columncontamination. As will be appreciated by one of ordinary skill in theart after reading this specification, one such sample introductiontechnique can be effected by butt-coupling an associated rotor to astator (not shown) on the external surface of a miniaturized columnwhere the rotor selectively interfaces external tubing and fluid sourceswith the inlet port 18 and apertures 26 and 28. In this manner, therotor allows a sample to be flushed from the by-pass channel 24 intoexternal tubing--from which tubing the sample can then be introducedinto the column via the inlet port 18 for liquid phase analysis thereof.Thus, a miniaturized column device formed in a polyimide substrateenables a ceramic rotor--pressed to the device using tensioned force (toform a liquid-tight seal)--to still rotate between selected aperturepositions on the device due to the friction characteristics of the twomaterials. Other suitable rotors can be formed in rigid materials suchas, but not limited to, glass and non-conductive substrates.

Accordingly, in the practice of the invention, external hardwareprovides the mechanical valving necessary for communication of aminiaturized column device to different external liquid reservoirs, suchas an electrolyte solution, flush solution or the sample vialaser-ablated holes designed into the cover plate 12. This featureallows a variety of injection methods to be adapted to a miniaturizedplanar column device, including pressure injection, hydrodynamicinjection or electrokinetic injection. In the particular embodiment ofFIGS. 1A, 2 and 3, the external valving and injection means cancommunicate with the separation device by butt-coupling to thelaser-ablated apertures; however, any other suitable method ofconnection known in the art can be readily adapted to the invention.Further, it is noted that numerous other sample introduction and fluidinterfacing designs can be practiced and still fall within the spirit ofthe subject invention.

Referring still to FIGS. 1A and 2-4, a wide variety of means forapplying a motive force along the length of the separation compartment14 can be associated with the subject device. Particularly, a pressuredifferential or electric potential can be applied along the entirelength of the separation compartment by interfacing motive means withthe inlet port 18 and outlet port 22 using techniques well known in theart.

The use of substrates such as polyimides in the construction ofminiaturized columns herein allows the possibility of usingrefractive-index (RI) detection to detect separated analytes of interestpassing through the subject columns. In this manner, the provision of anassociated laser diode which emits radiation at a wavelength wherepolyimide is "transparent" (such as at >500 nm) allows for a detectionsetup where no additional features need to be ablated in the columndevices.

Referring particularly to FIGS. 2-4, in another preferred embodiment ofthe invention, one or more detection means can optionally be ablatedinto the substrate 4 and/or cover plate 12. Preferably, the detectionmeans will be disposed substantially downstream of the first end 16 ofthe separation compartment 14 to enable detection of separated analytesfrom the liquid sample. More specifically, an aperture 34 can be ablatedthrough substrate 4 to communicate with the separation compartment 14. Acorresponding aperture 36 can likewise be formed in cover plate 12, andarranged so that it will be in coaxial alignment with aperture 34 whenthe cover plate is affixed to the substrate to form the separationcompartment 14. In this manner, electrodes (not shown) can be connectedto the miniaturized column device via the apertures 34 and 36 to detectseparated analytes of interest passing through the separationcompartment by electrochemical detection techniques.

Referring now to FIG. 5, a related aspect of the invention is shown,comprising a miniaturized column device 2", wherein the column portionand the cover plate portion are formed in a single, flexible substrateindicated at 44. The flexible substrate 44 thus includes first andsecond portions, 44A and 44B, respectively, wherein each portion has asubstantially planar interior surface. First and second portions 44A and44B are separated by at least one fold means 46, such that the firstportion can be readily folded to overlie the second portion. Inparticularly preferred embodiments, fold means 46 can comprise a row ofspaced-apart perforations ablated in the flexible substrate,spaced-apart slot-like depressions or apertures ablated so as to extendonly part way through the substrate, or the like. The perforations ordepressions can have circular, diamond, hexagonal or other shapes thatpromote hinge formation along a predetermined straight line.

Thus, the miniaturized column device 2" is formed by first laserablating a microchannel 10' in the second substrate portion 44B. Aseparation compartment is then provided by folding the flexiblesubstrate 44 at the fold means 46 such that the first portion 44A coversthe microchannel 10' to form a separation compartment as describedabove.

In this manner, the accurate alignment of various component parts isreadily enabled by the provision of fold means 46. More particularly,such fold means 46 allows the first and second portions 44A and 44B tohingably fold upon each other to accurately align components which havebeen ablated in the first and second portions. In one particularembodiment, a detection means 48 is provided which is formed by ablatingan aperture in the first portion 44A of the flexible substrate. Thedetection means 48 is arranged to communicate with the microchannel 10'when the portions 44A and 44B are folded upon each other. In a relatedembodiment, the detection means 48 is arranged to correspond withanother detection means 50, comprising an aperture which has been laserablated in the second portion 44B to communicate with the microchannel10'. Thus, when the portions 44A and 44B are folded upon each other, acoaxial detection path is provided by the alignment of the correspondingdetection means 48 and 50. In this manner, the coaxially aligneddetection means enable optical detection of separated analytes passingthrough separation compartment via transmission of radiation orthogonalto the major axis of the separation compartment (and, accordingly,orthogonal to the direction of electro-osmotic flow in anelectrophoretic separation).

In yet further related aspects of the invention, optionalmicro-alignment means--formed either by laser ablation techniques or byother methods of fabricating shaped pieces well known in the art--areprovided in the miniaturized column devices 2 and/or 2" of FIGS. 1A and5, respectively. More specifically, a plurality of correspondinglaser-ablated apertures (not shown) can be provided in either thesubstrate 4 and cover plate 12, or in the first and second flexiblesubstrate portions 44A and 44B. The subject apertures are arranged suchthat coaxial alignment thereof enables the precise alignment of thesubstrate 4 with the cover plate 12, or of the first and second flexiblesubstrate portions 44A and 44B to align various features such asdetection means with an ablated elongate bore. Such optional alignmentcan be effected using an external apparatus with means (such as pins)for cooperating with the coaxial apertures to maintain the components orportions in proper alignment with each other.

Referring now to FIG. 1B, a further embodiment of the invention,indicated at 2' is shown comprising a preferred detection meansindicated generally at 42. More particularly, a first transparent sheet38 is provided wherein the cover plate 12 is interposed between thefirst transparent sheet and substrate 4. A second transparent sheet 40is also provided wherein the second sheet is disposed over the secondplanar surface 8 of the substrate 4. In this manner, detection means 42allows optical detection of separated analytes passing through aseparation compartment--that has been formed by the combination ofmicrochannel 10 and cover plate 12--via transmission of radiationorthogonal to the major axis of the separation compartment (and,accordingly, orthogonal to the direction of electro-osmotic flow in anelectrophoretic separation). Further, in the practice of the invention,the transparent sheets can comprise materials such as quartz, diamond,sapphire, fused silica or any other suitable substrate which enableslight transmission therethrough.

The subject transparent sheets can be formed with just enough surfacearea to cover and seal the detection apertures 34 and 36, or thosesheets can be sized to cover up to the entire surface area of the columndevice. In this regard, additional structural rigidity is provided to acolumn device formed in a particularly thin substrate film, such as athin-film polyimide substrate, by employing a substantially coextensiveplanar sheet of, for example, fused silica.

Accordingly, the above described optical detection means 42 enablesadaptation of a variety of external optical detection means tominiaturized columns constructed according to the invention. Further,sealing of the transparent sheets 38 and 40 to the miniaturized columndevice 2' is readily enabled, for example, when substrate 4 and coverplate 12 are formed in polyimide materials which include a layer of athermal adhesive form of polyimide, since it is known thatquartz/Kapton® bonds formed using such adhesives are very resilient.Sealing of other preferred transparent sheet materials, such as diamond,sapphire or fused-silica to the subject device can be accomplished usingadhesion techniques well known in the art.

The capability of detecting with radiation over a range ofelectromagnetic wavelengths offers a variety of spectrophotometricdetection techniques to be interfaced with a miniaturized columnaccording to the invention, including, but not limited to, near IR,UV/Vis, fluorescence, refractive index (RI) and Raman.

Further, as will be readily appreciated, the use of optical detectionmeans comprising apertures ablated into the substrate and cover plateprovides great control over the effective detection pathlength in aminiaturized column device constructed herein. In this regard, thedetection pathlength will be substantially equal to the combinedthickness of the substrate 4 and the cover plate 12, and detection pathlengths of up to 250 μm are readily obtainable using the subjectdetection means 42 in thin-film substrates such as polyimides.

Referring now to FIG. 6, it can be seen that apertures 34 and 36 providean enlarged volume in separation compartment 14 at the point ofintersection with the detection means 42, where the enlarged volume willbe proportional to the combined thickness of substrate 4 and cover plate12. In this manner, sample plugs passing through separation compartment14 can be subject to untoward distortion as the plug is influenced bythe increased compartment volume in the detection area, especially wherethe combined thickness of the substrate and cover plate exceeds about250 μm, thereby possibly reducing separation efficiency in the device.

Accordingly, in the present invention wherein detection path lengthsexceeding 250 μm are desired, an alternative device embodiment isprovided having laser-ablated features on two opposing surfaces of asubstrate. More particularly, in FIGS. 7A and 7B, a further embodimentof a miniaturized column device is generally indicated at 52. Theminiaturized column comprises a substrate 54 having first and secondsubstantially planar opposing surfaces respectively indicated at 56 and58. The substrate 54 has a first microchannel 60 laser ablated in thefirst planar surface 56 and a second microchannel 62 laser ablated inthe second planar surface 58, wherein the microchannels can be providedin a wide variety of geometries, configurations and aspect ratios asdescribed above.

The miniaturized column device of FIGS. 7A and 7B further includes firstand second cover plates, indicated at 64 and 66 respectively, which, incombination with the first and second microchannels 60 and 62, definefirst and second elongate separation compartments when substrate 54 issandwiched between the first and second cover plates.

Referring still to FIGS. 7A and 7B, a plurality of apertures can belaser-ablated in the device to provide an extended separationcompartment, and further to establish fluid communication means. Moreparticularly, a conduit means 72, comprising a laser ablated aperture insubstrate 54 having an axis which is orthogonal to the first and secondplanar surfaces 56 and 58, communicates a distal end 74 of the firstmicrochannel 60 with a first end 76 of the second microchannel 62 toform an extended separation compartment.

Further, an aperture 68, laser ablated in the first cover plate 64,enables fluid communication with the first microchannel 60, and a secondaperture 70, laser ablated in the second cover plate 66, enables fluidcommunication with the second microchannel 62. As will be readilyappreciated, when the aperture 68 is used as an inlet port, and thesecond aperture 70 is used as an outlet port, a miniaturized columndevice is provided having a flow path extending along the combinedlength of the first and second microchannels 60 and 62.

In the embodiment of the invention as shown in FIGS. 7A and 7B, a widevariety of sample introduction means can be employed, such as thosedescribed above. External hardware can also be interfaced to the subjectdevice to provide liquid handling capabilities, and a variety of meansfor applying a motive force along the length of the separationcompartment can be associated with the device, such as by interfacingmotive means with the first and/or second apertures 68 and 70 asdescribed above.

Additionally, a variety of detection means are easily included in thesubject embodiment. In this regard, a first aperture 78 can be laserablated in the first cover plate 64, and a second aperture 80 canlikewise be formed in the second cover plate 66 such that the first andsecond apertures will be in coaxial alignment with conduit means 72 whenthe substrate 54 is sandwiched between the first and second coverplates. Detection of analytes in a separated sample passing through theconduit means is thereby easily enabled, such as by connectingelectrodes to the miniaturized column via apertures 78 and 80 anddetecting using electrochemical techniques.

However, a key feature of the laser-ablated conduit means 72 is theability to provide an extended optical detection path length of up to 1mm, or greater, without experiencing untoward sample plug distortion dueto increased separation compartment volumes at the point of detection.Referring to FIGS. 7A, 7B and 9, first and second transparent sheets,indicated at 82 and 84 respectively, can be provided such that the firstcover plate 64 is interposed between the first transparent sheet and thefirst planar surface 56, and the second cover plate 66 is interposedbetween the second transparent sheet and the second planar surface 58.The transparent sheets 82 and 84 can be selected from appropriatematerials such as quartz crystal, fused silica, diamond, sapphire andthe like. Further, the transparent sheets can be provided having justenough surface area to cover and seal the apertures 78 and 80, or thosesheets can be sized to cover up to the entire surface area of the columndevice. As described above, this feature allows additional structuralrigidity to be provided to a column device formed in a particularly thinsubstrate.

As best shown in FIG. 9, the subject arrangement allows opticaldetection of sample analytes passing through the miniaturized columndevice to be carried out along an optical detection path length 86corresponding to the major axis of the conduit means 72. As will bereadily appreciated, the optical detection path length 86 issubstantially determined by the thickness of the substrate 54, and,accordingly, a great deal of flexibility in tailoring a miniaturizedcolumn device having a-meter column dimensions and optical path lengthsof up to 1 mm or greater is thereby enabled herein. In this manner, awide variety of associated optical detection devices can be interfacedwith the novel miniaturized columns, and detection of analytes insamples passing through the conduit means 72 can be readily carried outusing UV/Vis, fluorescence, refractive index (RI), Raman and likespectrophotometric techniques.

Referring now to FIGS. 8A and 8B, a related embodiment of the inventionis shown, comprising a miniaturized column device 52', wherein thecolumn portion and the first and second cover plates are formed in asingle, flexible substrate generally indicated at 88. The flexiblesubstrate 88 thus comprises three distinct regions, a column portion88B, having first and second substantially planar opposing surfaces 56'and 58', respectively, where the column portion is interposed between afirst cover plate portion 88A and a second cover plate portion 88C. Thefirst and second cover plate portions have at least one substantiallyplanar surface. The first cover plate portion 88A and the column portion88B are separated by at least one fold means 90 such that the firstcover plate portion can be readily folded to overlie the firstsubstantially planar surface 56' of the column portion 88B. The secondcover plate portion 88C and the column portion 88B are likewiseseparated by at least one fold means 92 such that the second cover platecan be readily folded to overlie the second substantially planar surface58' of the column portion 88B. In particularly preferred embodiments,each fold means 90 and 92 can comprise a row of spaced-apartperforations ablated in the flexible substrate, spaced-apart slot-likedepressions or apertures ablated so as to extend only part way throughthe substrate, or the like. The perforations or depressions can havecircular, diamond, hexagonal or other shapes that promote hingeformation along a predetermined straight line.

Thus, the miniaturized column device 52' is formed by laser ablating afirst microchannel 60' in the first planar surface 56' of the columnportion 88B, and a second microchannel 62' in the second planar surface58' of the column portion. Each microchannel can be provided in a widevariety of geometries, configurations and aspect ratios. A firstseparation compartment is then formed by folding the flexible substrate88 at the first fold means 90 such that the first cover plate portion88A covers the first microchannel 60' to form an elongate separationcompartment. A second separation compartment is then provided by foldingthe flexible substrate 88 at the second fold means 92 such that thesecond cover plate portion 88C covers the second microchannel 62' toform a separation compartment as described above. A conduit means 72',comprising a laser ablated aperture in the column portion 88B having anaxis which is orthogonal to the first and second planar surfaces 56' and58', communicates a distal end of the first microchannel 60' with afirst end of the second microchannel 62' to form a single, extendedseparation compartment.

Further, an aperture 68', laser ablated in the first cover plate portion88A, enables fluid communication with the first microchannel 60', and asecond aperture 70', laser ablated in the second cover plate portion88C, enables fluid communication with the second microchannel 62'. Asdescribed above, when the first and second apertures are used as aninlet and outlet port, respectively, a miniaturized column device isprovided having a flow path extending along the combined length of thefirst and second microchannels.

Detection means can optionally be included in the device of FIGS. 8A and8B. In one particular embodiment, a first aperture 78 can be laserablated in the first cover plate portion 88A, and a second aperture 80'can likewise be formed in the second cover plate portion 88C, whereinthe apertures are arranged to coaxially communicate with each other andcommunicate with the conduit means 72' when the flexible substrate 88 ishingably folded as described above to accurately align the apertures 78'and 80' with the conduit means 72'.

In yet further related aspects of the invention, optionalmicro-alignment means--formed either by laser ablation techniques or byother methods of fabricating shaped pieces well known in the art--areprovided in the miniaturized column device 52'. More specifically, aplurality of corresponding laser-ablated apertures (not shown) can beprovided in the column portion 88B and the first and second cover plateportions, 88A and 88C, respectively of the flexible substrate 88. Thesubject apertures are arranged such that coaxial alignment thereofenables the precise alignment of the column portion with one, or both ofthe cover plate portions to align various features such as the optionaldetection means with the ablated conduit. Such optional alignment can beeffected using an external apparatus with means (such as pins) forcooperating with the coaxial apertures to maintain the components areportions in proper alignment with each other.

Accordingly, novel miniaturized column devices have been described whichare laser ablated into a substrate other than silicon or silicon dioxidematerials, and which avoid several major problems that have come to beassociated with prior attempts at providing micro-column devices. Theuse of laser ablation techniques in the practice of the inventionenables highly symmetrical and accurately defined micro-column devicesto be fabricated in a wide class of polymeric and ceramic substrates toprovide a variety of miniaturized liquid-phase analysis systems.Particularly, miniaturized columns are provided which havemicro-capillary dimensions (ranging from 20-200 μm in diameter) andcolumn detection path lengths of up to 1 mm or greater. This feature hasnot been attainable in prior attempts at miniaturization, such as incapillary electrophoresis, without substantial engineering of a deviceafter capillary formation. Further, laser ablation of miniaturizedcolumns in inert substrates such as polyimides avoids the problemsencountered in prior devices formed in silicon or silicon dioxide-basedmaterials. Such problems include the inherent chemical activity and pHinstability of silicon and silicon dioxide-based substrates which limitsthe types of separations capable of being performed in those devices.

In the practice of the invention, miniaturized column devices can beformed by laser ablating a set of desired features in a selectedsubstrate using a step-and-repeat process to form discrete units. Inthis regard, a wide variety of devices can be laser ablated according tothe invention in condensation polymer substrates including polyimides,polyamides, poly-esters and poly-carbonates. Further, the invention canbe practiced using either a laser ablation process or a LIGA process toform templates encompassing a set of desired features, whereby multiplecopies of miniaturized columns can be mass-produced using injectionmolding techniques well known in the art. More particularly, multiplecopies of the novel miniaturized columns can be formed herein byinjection molding in substrates such as, but not limited to, thefollowing substrates: polycarbonates; polyesters, includingpoly(ethylene terephthalate) and poly(butylene terephthalate);polyamides, (such as nylons); polyethers, including polyformaldehyde andpoly(phenylene sulfide); polyimides, such as Kapton® and Upilex®;polyolefin compounds, including ABS polymers, Kel-F copolymers,poly(methyl methacrylate), poly(styrene-butadiene) copolymers,poly(tetrafluoroethylene), poly(ethylene-vinyl acetate) copolymers,poly(N-vinylcarbazole) and polystyrene.

Laser ablation of microchannels in the surfaces of the above-describedsubstrates has the added feature of enabling a wide variety of surfacetreatments to be applied to the microchannels before formation of theseparation compartment. That is, the open configuration of laser-ablatedmicrochannels produced using the method of the invention enables anumber of surface treatments or modifications to be performed which arenot possible in closed format constructions, such as in priormicro-capillaries. More specifically, laser ablation in condensationpolymer substrates provides microchannels with surfaces featuringfunctional groups, such as carboxyl groups, hydroxyl groups and aminegroups, thereby enabling chemical bonding of selected species to thesurface of the subject microchannels using techniques well known in theart. Other surface treatments enabled by the open configuration of theinstant devices include surface adsorptions, polymer graftings and thinfilm deposition of materials such as diamond or sapphire to microchannelsurfaces using masking and deposition techniques and dynamicdeactivation techniques well known in the art of liquid separations.

The ability to exert rigid computerized control over laser ablationprocesses enables extremely precise microstructure formation, which, inturn, enables the formation of miniaturized columns having featuresablated in two substantially planar components wherein those componentscan be aligned to define a composite separation compartment of enhancedsymmetry and axial alignment. Thus, in a further embodiment of theinvention, miniaturized column devices are provided wherein laserablation is used to create two component halves which, when folded oraligned with each other, define a single miniaturized column device.

Referring now to FIG. 10, a miniaturized column for liquid phaseanalysis of a sample is generally indicated at 102. The miniaturizedcolumn 102 is formed by providing a support body 104 having first andsecond component halves indicated at 106 and 108 respectively. Thesupport body can comprise a substantially planar substrate such as apolyimide film which is both laser ablatable and flexible so as toenable folding after ablation; however, the particular substrateselected is not considered to be limiting in the invention.

The first and second component halves, 106 and 108, each havesubstantially planar interior surfaces, indicated at 110 and 112respectively, wherein miniaturized column features can be laser ablated.More particularly, a first microchannel pattern 114 is laser ablated inthe first planar interior surface 110 and a second microchannel pattern116 is laser ablated in the second planar interior surface 112. Thefirst and second microchannel patterns are ablated in the support body104 so as to substantially provide the mirror image of each other.

Referring now to FIGS. 11 and 12, a separation compartment 118,comprising an elongate bore defined by the first and second microchannelpatterns 114 and 116 can be formed by aligning (such as by folding) thefirst and second component halves 106 and 108 in facing abutment witheach other. In the practice of the invention, the first and secondcomponent halves can be held in fixable alignment with one another toform a liquid-tight separation compartment using pressure sealingtechniques, such as by application of tensioned force, or by use ofadhesives well known in the art of liquid phase separation devices. Inone particular embodiment, first and second microchannels 114 and 116are provided having semi-circular cross-sections, whereby alignment ofthe component halves defines a separation compartment 118 having ahighly symmetrical circular cross-section to enable enhanced fluid flowtherethrough; however, as discussed above, a wide variety ofmicrochannel geometries are also within the spirit of the invention.

In a further preferred embodiment of the invention, the support body 104is formed from a polymer laminate substrate comprising a Kapton® filmco-extruded with a thin layer of a thermal plastic form of polyimidereferred to as KJ and available from DuPont (Wilmington, Del.). In thismanner, the first and second component halves 106 and 108 can be heatsealed together, resulting in a liquid-tight weld that has the samechemical properties and, accordingly, the same mechanical, electricaland chemical stability, as the bulk Kapton® material.

Referring now to FIGS. 10-12, the miniaturized column device 102 furtherincludes means for communicating associated external fluid containmentmeans (not shown) with the separation compartment 118 to provide aliquid-phase separation device. More particularly, a plurality ofapertures can be laser ablated in the support body 104, wherein theapertures extend from at least one exterior surface of the support bodyand communicate with at least one microchannel, said aperturespermitting the passage of fluid therethrough. More particularly, aninlet port 120 can be laser ablated in the first component half 106 tocommunicate with a first end 122 of the first microchannel 114. In thesame manner, an outlet port 124 can be ablated in the first componenthalf to communicate with a second end 126 of the first microchannel 114.

In this manner, a liquid phase separation device is readily provided,having a flow path extending from the first end 122 of the microchannel114 to the second end 126 thereof, by communicating fluids from anassociated source (not shown) through the inlet port 120, passing thefluids through the separation compartment 118 formed by the alignment ofmicrochannels 114 and 116, and allowing the fluids to exit theseparation compartment via the outlet port 126. Thus, a wide variety ofliquid phase analysis procedures can be carried out in the subjectminiaturized column device using techniques well known in the art.Furthermore, various means for applying a motive force along the lengthof the separation compartment 118, such as a pressure differential orelectric potential, can be readily interfaced to the column device viathe inlet and outlet ports, or by interfacing with the separationcompartment via additional apertures which can be ablated in the supportbody 104.

In particular preferred embodiments, the inlet port 120 can be formedsuch that a variety of external fluid and/or sample introduction meansare readily interfaced with the miniaturized column device 102. Asdiscussed above, such sample introduction means include externalpressure injection, hydrodynamic injection or electrokinetic injectionmechanisms.

Referring now to FIGS. 10 and 11, the miniaturized column device 102 canfurther include a detection means laser ablated in the support body 104.More particularly, a first aperture 128 is ablated in the firstcomponent half 106 to communicate with the first microchannel 114 at apoint near the second end 126 thereof. A second aperture 130 is likewiseformed in the second component half 108 to communicate with the secondmicrochannel 116. In this manner, a wide variety of associated detectionmeans can then be interfaced to the separation compartment 118 to detectseparated analytes of interest passing therethrough, such as byconnection of electrodes to the miniaturized column via the first andsecond apertures 128 and 130.

In yet a further preferred embodiment of the invention, an opticaldetection means is provided in the miniaturized column device 102. Inthis regard, first and second apertures 128 and 130 are ablated in thesupport body 104 such that when the component halves are aligned to formthe separation compartment 118, the apertures are in coaxial alignmentwith each other, wherein the apertures have axes orthogonal to the planeof the support body. As will be readily appreciated by one of ordinaryskill in the art, by providing transparent sheets (not shown)--disposedover the exterior of the support body 104 and covering the first andsecond apertures 128 and 130--a sample passing through the separationcompartment 118 can be analyzed by interfacing spectrophotometricdetection means with the sample through the transparent sheets usingtechniques well known in the art. The optical detection pathlength issubstantially determined by the combined thickness of the first andsecond component halves 106 and 108. In this manner, an opticaldetection pathlength of up to 250 μm is readily provided by ablating theminiaturized column device in a 125 μm polymer film.

Accordingly, there have been described several preferred embodiments ofa miniaturized column device formed according to the invention by laserablating microstructures in component parts and aligning the componentsto form columns having enhanced symmetries. As described above,formation of the subject microchannels in the open configuration enablesa wide variety of surface treatments and modifications to be applied tothe interior surfaces of the channels before alignment of the componentsto provide the separation compartment. In this manner, a wide variety ofliquid phase analysis techniques can be carried out in the compositeseparation compartments thus formed, including chromatographic,electrophoretic and electrochromatographic separations.

In yet a further embodiment of the invention, optional means areprovided for the precise alignment of component support body halves,thereby ensuring accurate definition of a composite separationcompartment formed thereby. More particularly, optional micro-alignmentmeans are provided to enable enhanced alignment of laser-ablatedcomponent parts, such as the precise alignment of complementarymicrochannels with each other, detection apertures with microchannels,inlet and outlet ports with microchannels, detection apertures withfurther detection apertures, and the like.

Referring now to FIGS. 13 and 14, a miniaturized column deviceconstructed according to the present invention is generally indicated at150, wherein the device is formed in a single flexible substrate 152.The column device comprises first and second support body halves,indicated at 154 and 156 respectively, each half comprising asubstantially planar interior surface indicated at 158 and 160respectively. The interior surfaces have laser-ablated microstructuresformed therein, generally indicated at 162, wherein the microstructuresare arranged to provide the mirror image of each other in the samemanner as described above.

More particularly, the accurate alignment of the component parts iseffected by forming a miniaturized column device in a flexible substrate152 having at least one fold means, generally indicated at 180, suchthat the first body half 154 can be folded to overlie the second bodyhalf 156. The fold means 180 can comprise a row of spaced-apartperforations ablated in the substrate 152, spaced-apart slot-likedepressions or apertures ablated so as to extend only part way throughthe substrate, or the like. The perforations or depressions can havecircular, diamond, hexagonal or other shapes that promote hingeformation along a predetermined straight line.

Accordingly, in the practice of the invention, the fold means 180 allowsthe first and second support body halves 154 and 156 to hingably foldupon each other to precisely align various composite features that aredefined by the microstructures ablated in the first and second planarinterior surfaces 158 and 160.

In a related embodiment, optionally micro-alignment means are providedin the first and/or second planar interior surfaces 158 and 160. Themicro-alignment means are formed either by laser ablation or by othermethods of fabricating shaped pieces well known in the art. Morespecifically, a plurality of laser-ablated apertures (not shown) can beprovided in the first and second support body halves 154 and 156,wherein the apertures are arranged such that the coaxial alignmentthereof effects the precise alignment of the support body halves todefine composite features, such as an ablated elongate bore. Suchalignment can be maintained using an external apparatus with means (suchas pins) for cooperating with the coaxial apertures to support the bodyhalves in proper alignment with one another.

Referring to FIGS. 13 and 14, in yet another particular embodiment ofthe invention, micro-alignment means can be formed in the first andsecond support body halves 154 and 156 using fabrication techniques wellknown in the art, e.g., molding or the like. In this manner, a pluralityof projections, indicated at 164, 166 and 168, can be formed in thefirst support body half 154. A plurality of depressions, indicated at170, 172 and 174, can be formed in the second support body half 156.

In this particular configuration, the micro-alignment means are designedto form corresponding (mating) structures with each other, wherebyprojection 164 mates with depression 170, projection 166 mates withdepression 172, and projection 168 mates with depression 174 when thesupport body halves are aligned in facing abutment with each other. Inthis manner, positive and precise alignment of support body halves 154and 156 is enabled, thereby accurately defining composite featuresdefined by the laser-ablated microstructures 162.

As will be readily apparent to one of ordinary skill in the art afterreading this specification, a wide variety of correspondingmicro-alignment features can be formed in the subject miniaturizedcolumn devices without departing from the spirit of the instantinvention. These additional features include any combination of holesand/or corresponding structures such as grooves and ridges in thecomponent parts where the features cooperate to enable precise alignmentof the component body parts.

In yet a further aspect of the invention, an electrical detection meansis provided that is capable of detecting changes in the electricalproperties of a liquid sample passing through the separation compartmentof any of the miniaturized column device embodiments of the invention.Referring to FIG. 15, a miniaturized column device constructed accordingto the invention is generally indicated at 202. The device is formed inan appropriate substrate 204 which has at least one substantially planarsurface, indicated at 206. A microchannel 208 is formed in the substrate206 as described above using laser ablation techniques. Thus, a coverplate 210 arranged over the microchannel 208 forms a separationcompartment. The cover plate can be formed from any suitable substratesuch as polyimide, where the selection is limited only by the avoidanceof undesirable separation surfaces such as silicon or silicon dioxidematerials.

Referring now to FIGS. 15-17, a plurality of detection means, comprisingfirst and second electrical communication paths 212 and 214,respectively, are arranged opposite each other relative to themicrochannel 208. More particularly, a first end 216 of communicationpath 212 is arranged alongside and directly adjacent to a first side ofthe microchannel 208. A first end 218 of communication path 214 isarranged alongside and directly adjacent to a second side of themicrochannel such that the ends 216 and 218 form a detection pathinterrupted by the microchannel. The communication path ends 216 and 218have no direct contact with the microchannel. More particularly, thereis at least about several μm of substrate 204 between the ends (216 and218) and the microchannel 208. In this manner, there is no directcontact of the communication paths with the sample passing through theseparation compartment. This arrangement avoids galvanic contact of thecommunication paths with the sample and associated electrolysis leadingto gas bubble generation, and ensures that the communication pathsremain stable and provide accurate, reproducible measurements.

Referring still to FIGS. 15-17, connection of the subject detectionmeans with an appropriate associated signal source, such as an AC signalsource (not shown), is effected through exposed contact points 220 and222, which are arranged at a second end of communication paths 212 and214, respectively. In this manner, communication paths 212 and 214 areused to provide an antenna circuit which is capable of generating anelectric field encompassing the separation compartment, whereby a phaseshift due to changes in the conductance or permittivity of a streamingliquid sample passing through the compartment provides a linear,detectable signal to an associated impedance meter or any otherappropriate permittivity detector. More particularly, the communicationpaths 212 and 214 can be used as electrode antennae to generate anelectric field, wherein the antennae form part of a resonance circuit.The amplitude of an oscillating frequency in the subject resonancecircuit will be proportional to the conductivity of the contents of theseparation compartment. Thus, a constant phase lag can be generated byvarying the frequency of an oscillating signal transmitted by theantennae. The phase lag will fluctuate in response to changes in theconductivity, permittivity, or both, of the contents of the separationcompartment streaming through the electric field which is in turn fedback to vary the frequency in the antennae to compensate for the shiftin the phase lag, thereby providing a detectable signal.

In one particular embodiment, communication paths 212 and 214 are formedon the substrate 204 by sputtering deposition techniques well known inthe art. In a related embodiment, the communication paths can be formedin copper-polymer laminate substrates, wherein the paths are formedusing etching, ablation or micromachining techniques well known in theart. In one particularly preferred embodiment, communication paths 212and 214 can be formed using laser ablation techniques, whereby the pathconfiguration is laser-ablated in the substrate to form depressionswhich can subsequently be filled with an appropriate conductivematerial. The electric field strength of antennae circuit formed by thepath configuration is controlled by the voltage applied thereto, and thedistance between the antennae. Referring particularly to FIG. 17, suchtechniques allow the formation of conductive paths in anylaser-ablatable configuration, and further readily enable the formationof paths having a height h, which is coextensive with the height h, ofthe microchannel 208. In this regard, the strength of a generatedelectric signal can be controlled by variations in the height and thelength of the opposing communication path ends 216 and 218.

A number of additional configurations of electrical detection means canbe provided in the above-described devices. Referring to FIGS. 18-20, afurther embodiment of the invention, comprising a miniaturized columndevice, is generally indicated at 252, having a cover plate 254 arrangedover a microchannel 256 to form a separation compartment. A plurality ofdetection means, comprising first and second electrical communicationpaths 258 and 260, respectively, are arranged in spaced-apart relationto each other in the longitudinal direction along a first side ofmicrochannel 256.

Referring particularly to FIGS. 19 and 20, communication paths 258 and260 have first ends 262 and 264, respectively, which are arrangeddirectly adjacent to the microchannel 256; however, ends 262 and 264 arenot in direct contact with the microchannel as described above. Exposedcontact points 266 and 268, respectively arranged at second ends ofcommunication paths 258 and 260, are available for connection to anassociated signal generator. In this manner, communication paths 212 and214 can be used herein to provide an antenna circuit which is capable ofgenerating an electric field encompassing the separation compartment,whereby a phase shift produced by changes in the conductance orpermittivity of a streaming liquid sample passing through thecompartment provides a detectable signal as described above. Referringparticularly to FIG. 20, communication paths 258 and 260 can be formedusing laser ablation techniques, such that the height h₃ of path ends262 and 264 is co-extensive with the height h₄ of microchannel 256 toprovide enhanced signal strength.

In yet a further related embodiment, an electrical detectionconfiguration is provided comprising a plurality of serially arrangedcoils arranged in coaxial relation about the separation compartment. Inone embodiment, a plurality of serially arranged annular coils areprovided to approximate an electrical coil detection configuration.Referring to FIGS. 21 and 22, a miniaturized column device 282 isdepicted, having a microchannel 284 laser-ablated in a firstsubstantially planar surface 286 of an appropriate substrate 288. Themicrochannel 284, in combination with a cover plate 290, forms aseparation compartment 292 as described above. Referring particularly toFIGS. 22 and 24, a plurality of detection means, comprising first 294and second 296 communication paths, are provided, having first andsecond annular coil portions 298 and 300, respectively, that arearranged in coaxial relation about the separation compartment 292 and inspaced-apart relation to each other in the longitudinal direction alongthe compartment. Exposed contact points 302 and 304, arranged at distalends of communication paths 294 and 296, respectively, allow connectionof the subject detection means with an appropriate signal source.

In this manner, electrical detection can be effected using techniqueswell known in the art. More particularly, a magnetic field can begenerated in the core of a first annular coil portion, e.g., 298. A partof the induced electrical field provided by that magnetic field travelsalong the separation compartment toward the second annular coil portion300. The second coil 300 is capable of detecting and measuring theelectrical field. Accordingly, a phase shift produced by changes in theconductance or permittivity of a streaming liquid sample passing throughthe compartment provides a detectable signal as described above.

Communication paths 294 and 296 can be formed herein using any suitablemethod, including sputtering or other depositions, laser ablation,etching or micromachining techniques. In one particular embodiment, theannular coil configuration of communication path 294 can be formed as acomposite structure. Referring to FIGS. 21, 23 and 25, a first portionof the annular coil configuration, indicated at 310, is provided by aconductive strip formed on a second planar surface 316 of substrate 288.The first portion 310 is thus arranged below microchannel 284. Secondand third portions, 312 and 314, are formed by laser-ablation, whereindepressions are ablated in substrate 288 to communicate with firstconductive portion 310, which depressions are subsequently filled-inwith a suitable conductive material. Portions 312 and 314 are thusarranged in opposing relation to each other on first and second sides,respectively, of microchannel 284.

In this manner, a substantially U-shaped configuration is provided whichsurrounds the microchannel without coming into direct contact with theinterior of the channel. The composite annular coil configuration iscompleted by forming a similar structure in cover plate 290, comprisinga complementary U-shaped conductive portion 318 that is disposed so asto contact portions 312 and 314 when cover plate 290 is arranged overmicrochannel 284 to form the separation compartment as described above.

In a further aspect of the invention, a number of further detectionconfigurations are provided, wherein integrated lightguides are employedto provide enhanced detection capabilities to the subject devices. Inthis regard, each of the miniaturized column device embodiments of thepresent invention can include means for employing one or more optionallightguide means.

More particularly, referring to FIG. 1A, a detection means comprising asingle aperture 36 formed in cover plate 12 and communicating withseparation compartment 14 will readily accept a lightguide means (notshown), such as an optical fiber, integrated lens configuration, or thelike. In one particular embodiment, the lightguide means can comprise anoptical fiber that is selected to have substantially the same dimensionsas the aperture 36, whereby interface of the optical fiber with theaperture to communicate with the separation compartment provides aliquid-tight seal. The optical fiber can be configured so as to transmita fluorescent excitation wavelength into the separation compartment 14,and to receive a fluorescent emission signal therefrom. In this manner,fluorescence detection techniques can be carried out using methods thatare well known in the art.

Referring still to FIG. 1A, a further detection meansconfiguration--comprising aperture 36 which is coaxially aligned with asecond aperture 34 formed in substrate 4--provides an optical detectionpath. The optical detection path thus formed allows detection ofseparated analytes passing through separation compartment 14 viatransmission of radiation orthogonal to the major axis of the separationcompartment. Thus, a plurality of optional lightguide means, such asoptical fibers and/or integrated lens means, can be readily interfacedwith apertures 34 and 36 to communicate with the separation compartment14 as previously described. In one particular embodiment, a first suchoptical fiber can be employed for sample illumination, and a second forlight collection to enable near IR or UV/Vis optical detection ofseparated analytes passing through the separation compartment.

In further related embodiments, each of the column device embodiments ofthe present invention which include either a single aperturecommunicating with a separation compartment, or a plurality of coaxiallyaligned apertures communicating with a separation compartment andforming an optical detection path, can accommodate one or more optionallightguide means as just described.

Referring now to FIG. 26, yet a further related embodiment of theinvention is shown, comprising a miniaturized column device generallyindicated at 352. The device is formed from a first substrate portion354, having a substantially planar surface 356 with a first microchannel358 laser-ablated therein. The column device further includes a secondsubstrate portion 360, having a substantially planar surface 362 with asecond microchannel 364 laser-ablated therein. The second microchannel364 is arranged to provide the mirror image of the first microchannel358 when the first and second substantially planar surfaces 356 and 362are arranged in facing abutment with each other to form a separationcompartment as described above.

The device 352 further comprises a detection means formed from alaser-ablated groove set. More particularly, a first groove 366 islaser-ablated in the first planar surface 356 to communicate with thefirst microchannel 358. A second, complementary laser-ablated groove368--which communicates with the second microchannel 364--is arranged inthe second planar surface 362 to provide the mirror image of the firstgroove 366. In this manner, a detection path communicating with theseparation compartment is provided. The detection path comprises acompartment that is formed from the complementary groove set when thefirst and second substantially planar surfaces 356 and 362 are arrangedin facing abutment with each other.

Referring now to FIGS. 26 and 27, the detection path is configured toreadily accept an associated lightguide means. Thus lightguide means370, comprising an optical fiber, integrated lens configuration, or likemeans can be disposed within the detection path to communicate with theseparation compartment. In one particular embodiment, lightguide means370 comprises an optical fiber that is selected to have substantiallythe same dimensions as the compartment formed by the complementarygroove set 366 and 368, whereby insertion of the optical fiber withinthe compartment provides a liquid-tight seal. The lightguide means 370can thus be configured so as to transmit a fluorescent excitationwavelength into the separation compartment, and to receive a fluorescentemission signal therefrom as described above.

Referring now to FIG. 28, a related embodiment of the invention is showncomprising a miniaturized. column device generally indicated at 382. Thedevice is formed in a selected substrate 384 having a substantiallyplanar surface 386. A microchannel 388 is laser-ablated in the planarsurface 386 which is in communication with first and secondlaser-ablated grooves, indicated at 390 and 392, respectively. The firstand second grooves are arranged opposite each other relative to themicrochannel 388, thereby forming a detection path when cover plate 394is arranged over the planar surface 386 to form a separation compartmentas previously described.

Referring now to FIGS. 28 and 29, the optical detection path thus formedallows detection of separated analytes passing through the separationcompartment via transmission of radiation orthogonal to the major axisof the separation compartment. Thus, first and second lightguide means,respectively indicated at 394 and 396, and comprising optical fibers,integrated lens means or the like, can be readily disposed withingrooves 390 and 392 to communicate with the separation compartment aspreviously described. In one particular embodiment, a first opticalfiber 394 can be employed for sample illumination, and a second fiber396 for light collection to enable near IR or UV/Vis optical detectionof separated analytes passing through the separation compartment.

In a further aspect of the invention, miniaturized column devices formedaccording to the invention are provided having a plurality of detectionmeans that converge at a particular location in the separationcompartment. Referring to FIG. 30, one such device is generallyindicated at 402. The column device comprises a laser-ablatedmicrochannel 404 formed in a substantially planar surface 406 of asuitable substrate 408. Microchannel 404, in combination with coverplate 410 provides an elongate separation compartment 412. A firstdetection path, generally indicated at 414, is formed by the coaxialalignment of aperture 416--which is laser-ablated in cover plate 410 andarranged to communicate with separation compartment 412--and aperture418, which is laser-ablated in substrate 408 to communicate with theseparation compartment 412.

A second detection path, generally indicated at 420, is provided byfirst and second laser-ablated grooves, indicated at 422 and 424,respectively. The grooves are formed in planar surface 406 tocommunicate with the separation compartment 412 at first and secondopposing sides thereof. In this manner, first and second grooves 422 and424 are arranged opposite each other relative to the separationcompartment 412 and form a second detection path when cover plate 410 isarranged over planar surface 406 to provide the separation compartmentas previously described.

The first and second detection paths, 414 and 420, provide two mutuallyperpendicular optical axes which intersect within the separationcompartment, wherein those axes are also orthogonal to the major axis ofthe separation compartment 412. Thus, a wide variety of simultaneousdetection techniques can be carried out at the intersection of detectionpaths within the separation compartment.

In one particular embodiment, a first transparent sheet (not shown) canbe arranged over aperture 416, and a second transparent sheet (notshown) can likewise be arranged over aperture 418 wherein saidtransparent sheets, in combination with the first detection path 414form an optical detection path. In another embodiment, first and secondlightguide means (not shown) can be interfaced with first and secondapertures 416 and 418 to communicate with the separation compartment412. As described above, such lightguides can comprise optical fibersthat are capable of sample illumination and light collection to enablenear IR or UV/Vis optical detection of separated analytes passingthrough the separation compartment.

In the above-described devices, the second detection path 420 canreadily accommodate optional lightguide means to provide simultaneousoptical detection, electrode pairs to provide simultaneouselectrochemical detection, or communication paths to provide forsimultaneous electrical detection, each of which detectionconfigurations has been previously described.

In FIG. 31, yet a further related embodiment is shown, comprising aminiaturized column device generally indicated at 452. The devicecomprises a separation compartment 454 formed from a laser-ablatedmicrochannel 456 in a planar surface of a suitable substrate 458 and acover plate 460. A first detection means comprising a detection path 462is formed by the coaxial alignment of aperture 464 in cover plate 460and aperture 466 in substrate 458 wherein each said aperturecommunicates with the separation compartment 454 as described above. Afurther detection means is formed from a laser-ablated groove 468 insubstrate 458 which communicates with the separation compartment atsubstantially the same point that detection path 462 communicates withthe separation compartment to provide a detection intersection.

Thus, first and second transparent sheets can be arranged over apertures464 and 466, or lightguide means can be interfaced with said aperturesto form an optical detection path from detection path 462 as previouslydescribed. Such arrangements allow detection techniques such as near IRor UV/Vis to be used in the detection of separated analytes passingthrough the separation compartment 454.

The further detection means is configured to readily accept anassociated lightguide means. Thus, a lightguide means (not shown),comprising an optical fiber, integrated lens configuration, or likemeans, can be disposed within laser-ablated groove 468 to communicatewith the separation compartment. In one particular embodiment, thelightguide means comprises an optical fiber that is configured totransmit a fluorescent excitation wavelength into the separationcompartment, and to receive a fluorescent emission signal therefrom asdescribed above. The lightguide means can also be configured to onlyreceive light emission (e.g., fluorescence) in detections whereinexcitation is performed through aperture 466.

Further, while the present invention has been described with referenceto specific preferred embodiments, it is understood that the descriptionand examples included herein are intended to illustrate and not limitthe scope of the invention, which is defined by the scope of theappended claims.

What is claimed is:
 1. A method of forming a plurality of identical miniaturized column devices, comprising the steps of:(a) providing a master support body having a substantially planar interior surface, wherein the master support body is comprised of a material other than silicon and silicon dioxide; (b) forming a master copy of a miniaturized column device by: forming a microchannel directly in the interior surface of the master support body; (c) forming a mold insert by: (i) coating the interior surface of the master copy with a layer of a metal; (ii) depositing a metal thereon to fill the microchannel formed in the interior surface; and (iii) separating the mold insert from the master support body, whereby a mold insert is provided; and (d) preparing additional miniaturized column devices identical to the master copy by filling the mold insert with a suitable polymeric or ceramic material, whereby a miniaturized column device thus formed can be covered with a plate over the microchannel of the additional miniaturized column device to form a device having a microchannel for a fluid to pass therethrough.
 2. A method of forming a plurality of identical miniaturized channel devices, comprising the steps of:(a) providing a master support body having a substantially planar surface, wherein the master support body is comprised of a material other than silicon and silicon dioxide; (b) forming a master copy of a miniaturized channel device by forming a microchannel directly in the surface of the master support body; (c) forming a mold insert by filling the microchannel formed in the surface of the master support body with metal and separating the mold insert from the master support body, the mold insert having a first substantially planar surface corresponding to the substantially planar surface of the master support body; (d) preparing additional miniaturized channel devices identical to the master copy by filling the mold insert with a suitable polymeric or ceramic material; and (e) removing each replica from the mold insert and arranging a plate on the first planar surface of each replica, to form each miniaturized channel device; wherein each miniaturized channel device comprises a substrate comprising a replica and having first and second substantially planar surfaces, said substrate having a microchannel in the first planar surface and a plate arranged over the first planar surface, said plate in combination with the microchannel defining an elongate compartment for fluid to pass therethrough.
 3. The method of claim 2 further comprising molding replicas of a plate having a microchannel thereon and arranging a replica of said plate on the first planar surface of each replica formed using the mold insert.
 4. The method of claim 2 further comprising forming a microchannel on another planar surface on the opposite side of the substantially planar surface of the master support body, forming a mold insert using the master support body; preparing replicas of the master support body using the mold insert; and arranging a plate on each side of each replica of the master support body to form each miniaturized channel device.
 5. The method of claim 2 further comprising forming a coat of metal on the substantially planar surface of the master copy with a metal and depositing metal on said coat of metal to fill the microchannel to form the mold insert.
 6. The method of claim 2 further comprising coating the substantially planar surface of the master copy with a layer of a first metal and depositing a second metal thereon to fill the microchannel.
 7. The method of claim 2 further comprising forming a plurality of apertures in the master support body, said apertures being open for fluid communication with the microchannel.
 8. The method of claim 2 further comprising forming the microchannel by laser ablation.
 9. A method of forming a plurality of identical miniaturized channel devices, comprising the steps of:(a) providing a master support body having first and second halves, said halves comprising substantially planar surfaces for forming microchannels thereon, said planar surfaces comprising interior surfaces, wherein the master support body is comprised of a material other than silicon and silicon dioxide; (b) forming a master copy of a miniaturized channel device by: (i) forming a first microchannel directly in the interior surface of the first half of the master support body; and (ii) forming a second microchannel directly in the interior surface of the second half of the master support body, wherein the second microchannel is arranged to provide the mirror image of the first microchannel; (c) forming a mold insert from each half of the master support body by filling the microchannel formed in the interior surface with metal and separating the mold insert from each half of the master support body, whereby a mold insert is provided for each half; (d) preparing additional miniaturized channel devices identical to the master copy by filling the mold inserts with a suitable polymeric or ceramic material; and (e) removing each replica from the mold insert and arranging the first half on the second half of each replica, to form each miniaturized channel device; wherein each miniaturized channel device comprises a first half and a second half each having a microchannel facing the other, said microchannel in the first half in combination with the microchannel in the second half defining an elongate compartment for the passage of a fluid therethrough. 